Kengo Kaneko

Bradykinin enhances GLUT4 translocation through the increase of insulin receptor tyrosine kinase in primary adipocytes: Evidence that bradykinin stimulates the insulin signalling pathway

SummaryIt has been suggested that bradykinin stimulates glucose uptake in experiments in vivo and in cultured cells. However, its mechanism has not yet been fully elucidated. In this study, the effects of bradykinin on the insulin signalling pathway were evaluated in isolated dog adipocytes. The bradykinin receptor binding study revealed that dog adipocytes possessed significant numbers of bradykinin receptors (Kd=83 pmol/l, binding sites = 1.7×104 site/cell). Reverse transcription-polymerase chain reaction amplification showed the mRNA specific for bradykinin B2 receptor in the adipocytes. Bradykinin alone did not increase 2-deoxyglucose uptake in adipocytes; however, in the presence of insulin (10−7 mol/l) it significantly increased 2-deoxyglucose uptake in a dose-dependent manner. Bradykinin also enhanced insulin stimulated GLUT4 translocation from the intracellular fraction to the cell membrane, and insulin induced phosphorylation of the insulin receptor Β subunit and insulin receptor substrate-1 (IRS-1) without affecting the binding affinities or numbers of cell surface insulin receptors in dog adipocytes. The time-course of insulin stimulated phosphorylation of the insulin receptor Β subunit revealed that phosphorylation reached significantly higher levels at 10 min, and stayed at the higher levels until 120 min in the presence of bradykinin, suggesting that bradykinin delayed the dephosphorylation of the insulin receptor. It is concluded that bradykinin could potentiate insulin induced glucose uptake through GLUT4 translocation. This effect could be explained by the potency of bradykinin to upregulate the insulin receptor tyrosine kinase activity which stimulates phosphorylation of IRS-1, followed by GLUT4 translocation.

Impact of Natural IRS-1 Mutations on Insulin Signals: Mutations of IRS-1 in the PTB Domain and Near SH2 Protein Binding Sites Result in Impaired Function at Different Steps of IRS-1 Signaling

Insulin receptor substrate-1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates various insulin signals downstream. In this study, we have examined the impact of three natural IRS-1 mutations identified in NIDDM patients (G971R, P170R, and m209T) on insulin signaling. G971R is located near src homology 2 protein binding sites, and P170R and m209T are located in the phosphotyrosine binding domain of IRS-1. 32D-IR cells, stably overexpressing human insulin receptor, were transfected with wild-type human IRS-1 cDNA (WT) or three mutant IRS-1 cDNAs and analyzed. All the cell lines expressing mutant IRS-1 showed a significant reduction in ]3H]thymidine incorporation compared with WT. Upon insulin stimulation, cells expressing G971R showed a 39% decrease (P < 0.005) in phosphatidylinositol 3-kinase (PI 3-kinase) activity, a 43% decrease (P < 0.01) in binding of the 85-kDa regulatory subunit of PI 3-kinase, and a 22% decrease (P < 0.05) in mitogen-activated protein kinase activity compared with those expressing WT. Cells expressing P170R and m209T showed slight but significant decreases in PI 3-kinase activity (17 and 14%, respectively; both P < 0.05) and in binding of p85 (22 and 16%, respectively; both P < 0.05) and a greater decrease in mitogen-activated protein kinase activity (41 and 43%, respectively; both P < 0.005) compared with WT. After insulin stimulation, cells expressing P170R and m209T showed significant decreases in IRS-1 phosphorylation (37 and 42%, respectively; both P < 0.05) and in IRS-1 binding to the insulin receptor (48 and 53%, respectively; P < 0.01) compared with WT. G971R showed no changes in IRS-1 phosphorylation and in IRS-1 binding to the insulin receptor compared with WT. These data suggest that the impaired mitogenic response of P170R and m209T was mainly due to reduced binding to the insulin receptor, whereas the impaired response of G971R was mainly due to reduced association with PI 3-kinase p85.

Insulin inhibits glucagon secretion by the activation of PI3-kinase in In-R1-G9 cells

Intracellular mechanisms through which insulin inhibits glucagon secretion remain to be elucidated in glucagon secreting cells. In this study, we confirmed that, in In-R1-G9 cells, a pancreatic alpha cell line, insulin stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and activated phosphatidylinositol 3-kinase (PI3-kinase). We further studied, using wortmannin, an inhibitor of PI3-kinase, whether the inhibitory effect of insulin on glucagon secretion was mediated through PI3-kinase pathway in these cells. In static incubation studies, insulin significantly inhibited glucagon secretion at 2, 6 and 12 h, which was completely abolished by pretreatment with wortmannin. In perifusion studies, insulin significantly suppressed glucagon secretion after 10 min, which was also blocked by wortmannin. Insulin also reduced glucagon mRNA at 6 and 12 h but not at 2 h. Wortmannin also abolished insulin-induced reduction of glucagon mRNA. Insulin increased the amount of 85 kDa subunit of PI3-kinase in plasma membrane fraction (PM), with a reciprocal decrease of the kinase in cytosol fraction (CY). Insulin also increased PI3-kinase activity in PM, but not in CY. Our results suggest that insulin suppressed glucagon secretion by inhibiting glucagon release and gene expression. Both actions were mediated by activation of PI3-kinase. Recruitment and activation of PI3-kinase in plasma membrane might be relevant at least in part to insulin-induced inhibition of glucagon release.

Cis-acting DNA elements of mouse granulocyte/macrophage colony-stimulating factor gene responsive to oxidized low density lipoprotein.

We previously demonstrated that the induction of granulocyte/macrophage colony-stimulating factor (GM-CSF) played an important role in oxidized low density lipoprotein (Ox-LDL)-induced macrophage growth as a growth priming factor. The present study was undertaken to elucidate the transcriptional regulation of the GM-CSF gene using Raw 264.7 cells, a mouse macrophage cell line. Transient transfection into Raw 264.7 cells of several 5′-flanking regions of GM-CSF gene-luciferase fusion plasmids revealed the presence of two positive regulatory sites in regions spanning from −97 to −59 and from −59 to −37 and one negative regulatory site from −120 to −97 in unstimulated cells. When cells were stimulated by Ox-LDL, there was one positive responsive site from −225 to −120 and one negative responsive site from −97 to −59, which contained the NF-κB binding site. Computer analysis revealed the presence of a putative AP-2 binding site from −169 to −160. Mutagenesis of a putative AP-2 binding site and tandem repeat of this site in plasmid resulted in a complete loss and increased responsiveness to Ox-LDL, respectively. Electrophoretic mobility shift assay showed that Ox-LDL increased the binding of certain nuclear protein(s) to a putative AP-2 binding site but decreased their binding to NF-κB binding site. Supershift assay showed that nuclear proteins bound to NF-κB binding site contained, at least, p50 and p65 but could not demonstrate nuclear protein(s) bound to a putative AP-2 binding site. Our results suggested that a putative AP-2 binding site from −169 to −160 was a positive responsive element to Ox-LDL and that the NF-κB binding site from −91 to −82 was a negative responsive element in Ox-LDL-induced GM-CSF transcription.

Cell-Specific Regulation of IRS-1 Gene Expression: Role of E Box and C/EBP Binding Site in HepG2 Cells and CHO Cells

Insulin receptor substrate 1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream. We have previously shown that the levels of IRS-1 mRNA varied in different tissues. To elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and transacting factors in CHO and HepG2 cells. Using the chloramphenicol acetyltransferase (CAT) assay with the various deletion mutants of the IRS-1 promoter–CAT fusion plasmids, several regions responsible for positive or negative regulation in each cell line were identified. A region from −1645 to −1585 bp, which regulated expression negatively in CHO cells and positively in HepG2 cells, was further analyzed. Within this region sa fragment from −1645 to −1605 bp upregulated the IRS-1 promoter only in HepG2 cells, whereas a fragment from −1605 to −1585 bp downregulated only in CHO cells. In the gel mobility shift assay, several nuclear proteins that bind to these fragments were detected, and among them, two nuclear proteins that bind to a potential E box (nucleotide [nt] −1635 to −1630) and two nuclear proteins that bind to a potential C/EBP binding site (nt −1599 to −1591) were identified in HepG2 and CHO cells, respectively. CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell-specific regulation of the IRS-1 gene. We therefore concluded that the two nuclear proteins that bind to the E box regulate IRS-1 gene expression positively in HepG2 cells and the two nuclear proteins that bind to the C/EBP binding site regulate it negatively in CHO cells.

Cellular characterization of pituitary adenoma cell line (AtT20 cell) transfected with insulin, glucose transporter type 2 (GLUT2) and glucokinase genes: insulin secretion in response to physiological concentrations of glucose.

Summary We investigated the mechanisms of insulin secretion by transfecting into a pituitary adenoma cell line (AtT20) a combination of genes encoding human insulin (HI), glucose transporter type 2 (GLUT2) and glucokinase (GK), followed by studying the characteristics of these cells. In static incubation, a cell line transfected with insulin gene alone (AtT20HI) secreted mature human insulin but this was not in a glucose-dependent manner. Other cell lines transfected with insulin and GLUT2 genes (AtT20HI-GLUT2–3) or with insulin and GK genes (AtT20HI-GK-1) secreted insulin in response to glucose concentrations of only less than 1 mmol/l. In contrast, cell lines transfected with insulin, GLUT2 and GK genes (AtT20HI-GLUT2-GK-6, AtT20HI-GLUT2-GK-7 and AtT20HI-GLUT2-GK-10) showed a glucose-dependent insulin secretion up to 25 mmol/l glucose. Glucose utilization and oxidation were increased in AtT20HI-GLUT2-GK cell lines but not in AtT20HI, AtT20HI-GLUT2–3 and AtT20HI-GK-1 cells at physiological glucose concentrations, compared with AtT20 cells. Diazoxide, nifedipine and 2-deoxy glucose suppressed (p < 0.05) glucose stimulated insulin secretion in AtT20HI-GLUT2-GK-6 cells. Glibenclamide, KCl or corticotropin releasing factor (CRF) stimulated (p < 0.05) insulin secretion both in AtT20HI and AtT20HI-GLUT2-GK-6 cells. Insulin secretion stimulated by glibenclamide, KCl or CRF was further enhanced by the addition of 25 mmol/l glucose in AtT20HI-GLUT2-GK-6 cells but not in AtT20HI cells. In perifusion experiments, a stepwise increase in glucose concentration from 5 to 25 mmol/l stimulated insulin secretion in AtT20HI-GLUT2-GK cell lines but the response lacked a clear first phase of insulin secretion. Our results suggest that both GLUT2 and glucokinase are necessary for the glucose stimulated insulin secretion in at least rodent cell lines, and that other element(s) are necessary for a biphasic insulin secretion typically observed in beta cells. [Diabetologia (1998) 41: 1492–1501]

Bradykinin enhances insulin receptor tyrosine kinase in 32D cells reconstituted with bradykinin and insulin signaling pathways

We have previously shown that bradykinin potentiated insulin-induced glucose uptake through GLUT4 translocation in canine adipocytes and skeletal muscles. The aim of this study was to determine the molecular mechanism of bradykinin enhancement of the insulin signal. For this purpose, 32D cells, which express a limited number of insulin receptors and lack endogenous bradykinin B2 receptor (BK2R) or insulin receptor substrate (IRS)-1 were transfected with BK2R cDNA and/or insulin receptor cDNA and/or IRS-1 cDNA, and analyzed. In 32D cells that expressed BK2R and insulin receptor (32D-BKR/IR), bradykinin alone had no effect on the phosphorylation of the insulin receptor, but it enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor. In 32D cells that expressed BK2R, insulin receptor and IRS-1 (32D-BKR/IR/IRS1), bradykinin also enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1. An increase in insulin-stimulated phosphorylation of IRS-1 by treatment with bradykinin in 32D-BKR/IR/IRS1 cell was associated with increased binding of 85 kD subunit of phosphatidylinositol 3 (PI 3)-kinase and increased IRS-1 associated PI 3-kinase activity. These effects of bradykinin were not observed in 32D cells which lack the expression of BK2R (32D-IR/IRS1) or insulin receptor (32D-BKR/IRS1). Furthermore, tyrosine phosphatase activity against insulin receptor beta-subunit in plasma membrane fraction of 32D-BKR/IR cells was significantly reduced by bradykinin, suggesting that the effect of bradykinin was in part mediated by inhibition of protein tyrosine phosphatase(s). Our results clearly demonstrated that bradykinin enhanced insulin-stimulated tyrosine kinase activity of the insulin receptor and downstream insulin signal cascade through the BK2R mediated signal pathway.

Group-II phospholipase A2 enhances oxidized low density lipoprotein-induced macrophage growth through enhancement of GM-CSF release

Inflammatory process plays an important role in the development and progression of atherosclerotic lesions. Recently, group-II phospholipase A(2) (PLA(2)), an inflammatory mediator, was reported to exist in human atherosclerotic lesions and to enhance the development of murine atherosclerotic lesions. Oxidized low density lipoprotein (Ox-LDL) stimulates the growth of several types of macrophages in vitro. Since proliferation of macrophages occurs in atherosclerotic lesions, it is possible to assume that the Ox-LDL-induced macrophage proliferation might be involved in the progression of atherosclerosis. In this study, the role of group-II PLA(2) in the Ox-LDL-induced macrophage growth was investigated using thioglycollate-elicited mouse peritoneal macrophages. Thioglycollate-elicited macrophages significantly expressed group-II PLA(2) and released it into the culture medium. The Ox-LDL-induced thymidine incorporation into thioglycollate-elicited macrophages was three times higher than that into resident macrophages, whereas under the same conditions, granulocyte/macrophage colony-stimulating factor (GM-CSF) equally induced thymidine incorporation into both types of macrophages. Moreover, the Ox-LDL-induced GM-CSF release from thioglycollate-elicited macrophages was significantly higher than that from resident macrophages. In addition, the Ox-LDL-induced thymidine incorporation into macrophages obtained from human group-II PLA(2) transgenic mice and the GM-CSF release from these cells were significantly higher than those from their negative littermates, and the Ox-LDL-induced thymidine incorporation into human group-II PLA(2) transgenic macrophages was significantly inhibited by a polyclonal anti-human group-II PLA(2) antibody. These results suggest that the expression of group-II PLA(2) in thioglycollate-elicited macrophages may play an enhancing role in the Ox-LDL-induced macrophage growth through the enhancement of the GM-CSF release.